The development of synthetic compounds that mimic the presence of viruses may result in potent novel candidate drugs for the treatment of viral infection and cancer. The immune system employs at least four members of the family of Toll-like receptors (TLR3, TLR7, TLR8 and TLR9) to detect viruses based on the presence of certain characteristics of viral nucleic acid [1]. By far the most information is available for TLR9 [2, 3]. TLR9 detects so-called CpG motifs within microbial DNA [4]. Synthetic oligonucleotides containing such CpG motifs (CpG ODN) have been extensively studied over the last 10 years, and the lead compound ODN 2006 (identical with ODN 7909, ProMune) is currently entering clinical phase III for the treatment of cancer. Although CpG ODN represent strong Th1 vaccine adjuvants and show excellent anti-tumor activity in murine models of cancer [5, 6], their application for the treatment of human disease is limited. In mice TLR9 is expressed on B cells and both myeloid and plasmacytoid dendritic cells, whereas in humans expression is restricted to B cells and PDC [7, 8]. As a consequence, in the absence of T cell help, in mice CpG ODN stimulate both IL-12 and IFN-α in mice, while in humans, CpG ODN stimulate only IFN-α.
Recent data suggest that this deficit of CpG ODN in humans can be overcome by RNA oligonucleotides. Unlike TLR9 detecting DNA, TLR3, TLR7 and TLR8 all recognize RNA. TLR3 binds to long double-stranded (ds) RNA [9] and therefore by definition can not be activated by short synthetic oligoribonucleotides (ORN). TLR7 detects short dsRNA (as used for siRNA) [10] and both long and short single-stranded (ss) RNA [10-12]. RNA recognition by TLR8 is limited to ssRNA. Both TLR7 and TLR8 are expressed in human myeloid cells and thus, ORNs that serve as ligands for TLR7 and TLR8 are excellent candidates for eliciting the desired IL-12 response which is missing when CpG ODN is used for stimulation in the human system.
While for TLR9 the optimal sequence motif (CpG motif) has been exactly defined [4, 13, 14], the situation is much less clear for TLR7 and TLR8. It has been suggested that the potency of RNA oligonucleotides depends on a high content of G and U. One group proposed that the presence of the UGUGU motif confers IFN-α-inducing activity to RNA oligonucleotides. Our group defined a 9mer sequence motif [10]. WO 03/086280 discloses that guanosine, particularly guanosine in combination with uracil, are natural ligands of TLR8. Additional TLR8 ligands disclosed in WO 03/086280 include nucleic acid molecules containing one or more copies of GUU, GUG, GGU, GGG, UGG, UGU, UUG, UUU, UUGUGG, UGGUUG, GUGUGU, and GGGUUU.
In none of the studies published to date efforts were undertaken to distinguish potentially distinct sequence requirements for TLR7 and TLR8. However, small molecules (nucleoside analogues) have been proposed that specifically activate TLR7 or TLR8 [15], supporting the idea that TLR7 and TLR8 may indeed have preferences for distinct RNA motifs.
It is an object of the present invention to identify RNA oligonucleotide motifs for stimulating an immune response, in particular, IL-12 induction. It is also an object of the present invention to identify ligands for activating TLR8. It is another object of the present invention to develop a method for determining the immunostimulatory activity, in particular, the IL-12-inducing activity, of a RNA oligonucleotide. It is yet another object of the present invention to develop a method for predicting the immunostimulatory activity, in particular, IL-12-inducing activity, of a RNA oligonucleotide. It is a further object of the invention to develop a method for designing and preparing RNA oligonucleotide having or lacking immunostimulatory activity, in particular, IL-12-inducing activity. It is also an object of the invention to provide RNA oligonucleotides having high immunostimulatory activity which can be used to induce an immune response, in particular, IL-12 production, in patients in need thereof. It is yet another object of the present invention to provide antisense RNA molecules that either have or lack immunostimulatory activity which can be used to treat disorders caused by the expression or overexpression of disease/disorder-related genes.